Method for monitoring the temperature of a biochemical reaction

ABSTRACT

A method of monitoring the temperature of a biochemical reaction such as an amplification reaction is described. The method comprises effecting the reaction in the presence of a fluorescently labeled temperature probe DNA sequence which comprises a double stranded region which denatures at a predetermined temperature, the fluorescent label of said temperature probe sequence being arranged so that a detectable signal occurs at the point at which denaturation of said region takes places; and monitoring fluorescence from said reaction mixture so as to determine when said predetermined temperature has been reached.

[0001] The present invention relates to a method of carrying out anamplification reaction and in particular a polymerase chain reaction(PCR) using an internal temperature control mechanism.

[0002] A common problem in biochemical reactions, in particularminiaturised biochemical reactions is controlling the temperature.Invasive temperature probes add to the thermal mass of the sample andincrease time constraints associated with heating and cooling. Aparticular example where such a problem occurs is with minaturisedamplification reactions such as the PCR reaction. In this reaction,cycling between various accurate temperatures is an essential element Inoutline, the procedure consists of the following steps, repeatedcyclically. Denaturation: A mixture containing the PCR reagents(including the DNA to be copied, the individual nucleotide bases(A,T,G,C), suitable primers and Dolymerase enzyme) are heated to apredetermined temperature to separate the two strands of the target DNA.

[0003] Annealing: The mixture is then cooled to another predeterminedtemperature and the primers locate their complementary sequences on theDNA strands and bind to them.

[0004] Extension: The mixture is heated again to a further predeterminedtemperature. The polymerase enzyme (acting as a catalyst) joins theindividual nucleotide bases to the end of the primer to form a newstrand of DNA which is complementary to the sequence of the target DNA,the two strands being bound together.

[0005] Any interference with the reaching the predetermined temperaturesas a result of the temperature measurement can present a significantproblem in terms of the success of the amplification reaction.

[0006] The applicants have found a way in which the temperature presentin a biochemical reaction can be monitored without the need for theapplication of temperature probes.

[0007] According to the present invention there is provided a method ofmonitoring the temperature of a biochemical reaction, said methodcomprising effecting the reaction in the presence of a fluorescentlylabelled temperature probe DNA sequence which comprises a doublestranded region which denatures at a predetermined temperature, thefluorescent label or said temperature probe sequence being arranged sothat the nature of the fluorescence changes at the point at whichdenaturation of the said region takes place; and monitoring fluorescencefrom said reaction mixture so as to determine when the saidpredetermined temperature has been reached.

[0008] The labelled temperature probe DNA sequence added to the reactionmixture in the method acts as a temperature probe allowing thetemperature of the reaction to be accurately set without requiringexternal temperature probes.

[0009] The temperature probe DNA sequence may comprise a double strandedDNA sequence, or it may be in the form of a single nucleic acid strand,end regions of which hybridise together so as to form a loop or“hairpin” structure.

[0010] Suitable fluorescent labels include intercalating dyes, which areinterposed between the strands of a double stranded region of a DNAsequence. When the double stranded DNA region containing theintercalating dye reaches the predetermined temperature, it will bedenatured, thus releasing the intercalating dye present between thestrands. At this point the fluorescence from the mixture will reducesignificantly, giving a readable signal.

[0011] The process using a double stranded DNA sequence as a temperatureprobe is illustrated diagrammatically in FIG. 1 hereinafter.

[0012] When intercalating dye (2) is added to a solution of doublestranded DNA (1), it becomes interposed between the strands. Theconcentration of the dye (2) in this way produces a recognisable signal.On heating of the DNA so that it is denatured, dye is released and thisevent can be witnessed. Cooling to a temperature at which the saidsequence will anneal again results in the intercalating dye becomingagain trapped between the strands (see FIG. 1).

[0013] Suitable intercalating dyes include SYBRGreen™, SYBRGold™ andethidium bromide or other commercially available dyes.

[0014] Alternatively, the fluorescent label used in the method of theinvention may utilise fluorescence resonance transfer (FRET) as thebasis of the signal. These labels utilise the transfer of energy betweena reporter and a quencher molecule. The reporter molecule is excitedwith a specific wavelength of light for which it will normally exhibit afluorescence emission wavelength. The quencher molecule is also excitedat this wavelength such that it can accept the emission energy of thereporter molecule by resonance transfer when they are in close proximity(e.g. on the same, or a neighbouring molecule). The basis of FRETdetection is to monitor the changes at reporter and quencher emissionwavelengths.

[0015] For use in the context of the present invention, the DNA sequenceused as a temperature probe can be provided with a reporter and aquencher molecule, arranged so that the hybridisation of the strandsalters the spatial relationship between the quencher and reportermolecules. Examples of such arrangements are illustrated in FIG. 2 andFIG. 3.

[0016]FIG. 2 illustrates an Example where the temperature probe sequenceis a single stranded “hairpin” type sequence (3), where the end portionshybridise together. A reporter molecule (4) is attached in the region ofeither the 5′ or the 3′ end of the sequence and a quencher molecule (5)is attached at the opposite end such that they are brought into closeproximity when the sequence is in the form of the loop. In thisarrangement FRET occurs and so fluorescent signal from the reportermolecule is reduced whilst the signal from the quencher (5) molecule isenhanced.

[0017] On denaturation however, the opposed end regions of the sequenceseparate so that the reporter and quencher molecules become spaced andso FRET no linger occurs. This changes the signals from the respectivemolecules and so this event can be detected.

[0018] Another arrangement is illustrated in FIG. 3. In this case, thereporter (4) and quencher molecules (5) are located on different strands(6, 7 respectively) of a DNA temperature probe sequence and are locatedsuch that on hybridization of the strands, they are brought into closeproximity to each other so that RET can occur.

[0019] Yet a further embodiment is illustrated in FIG. 4. In this case,an intercalating dye (2) is used as an element of the MET system. Aquencher molecule (5) which can absorb radiation from the dye may bearranged on a strand of the temperature probe sequence such that it canabsorb radiation from dye which is close proximity to on hybridizationof the strands. When the temperature probe sequence reaches atemperature at which it is denatured, the dye (2) is dispersed and sothe signal from the quencher molecule (5) changes.

[0020] This embodiment is advantageous in that only a single label needbe applied to the temperature probe sequence. Single labelled sequencesof this type are more economical to produce.

[0021] In yet a further embodiment (FIG. 5), the reporter (4) andquencher (5) molecules are positioned on two oligonucleotide strands (9and 10 respectively) which do not hybridise together. They are howeverdesigned so that in use, they hydridise to a DNA sequence present in thereaction mixture, which may be a plasmid (11), such that the reporter(4) and quencher (5) are brought into close proximity and FRET can occurbetween them, giving a recognisable signal.

[0022] The DNA sequence to which they bind may be part of the reactionsystem, for example where the reaction being monitored is a PCR reactionwherein the DNA sequence comprises or is part of the amplificationtarget sequence. Alternatively, the sequences may be added to thereaction in order to provide the basis for the temperature probe of theinvention.

[0023] The temperature probe sequence of the invention may be designedso that it denatures at any desired predetermined temperature. Forexample, the denaturation temperature of a sequence depends to someextent on its length. Longer sequences will denature or melt at highertemperatures. Furthermore, it is known that the bases C and G bindtogether more strongly than A and T. Therefore, the greater the higherthe content of the bases G and C contained within a sequence, the higherthe melting point of the sequence will be. This feature is illustratedin FIG. 5 which shows the melting temperature of a DNA sequence plottedagainst the percentage of and GC base pairs which are present within in.Thus, by adjusting the GC content, the temperature probe sequence may bedesigned so that, if desired, it also has a predetermined length.

[0024] The method of the invention is particularly applicable for use inamplification reactions such as the polymerase chain reaction (PCR). Inthis case, the temperature probe sequence of the invention is introducedinto the reaction vessel. Suitably the temperature probe sequence isdesigned such that it generates a detectable signal when it reaches theoptimum annealing temperature of the target DNA sequence as this isintermediate temperature is most difficult to set accurately inpractice. However, more than one such temperature probe sequence may beadded and arranged to provide appropriate and preferably differentsignals when the predetermined extension and/or denaturationtemperatures have been reached.

[0025] The invention will now be particularly described by way ofexample with reference to the accompanying diagrammatic drawings inwhich:

[0026]FIG. 1 illustrates the formation and use of a labelled temperatureprobe sequence for use in the method of the invention;

[0027] FIGS. 2 to 5 represent alternative embodiments of the labelledtemperature probe sequences of the invention and the denaturationthereof;

[0028]FIG. 6 illustrates a construct used in the examples hereinafter;and

[0029]FIG. 7 shows the melting temperature of plasmid constructs andinserts as measured using the method of the invention, as a function ofthe percentage GC content of the construct, where the lighter linerepresents the oligonucleotides and the darker line represents the−47/−48 amplicon from constructs.

EXAMPLE 1

[0030] Oligonucleotides, 60 base pairs in length, were designed byrandomly removing the letters G, A, T and C from a paper bagComplementary pairs of the thus formed random oligonucleotides weremixed together at a final concentration of 1 μM and 1:40,000 dilution ofSYBRGreen™ reference dye. The mixtures were then loaded intoLightCycler™ tubes and the temperature slowly raised from 40° C. to 110°C. The fluorescence at 520 nm was measured and was seen to drop off asthe temperature was raised. The differential of fluorescence was used todetermine the peak rate of change (i.e. drop) which corresponds to thestrands melting. 20%, 40%, 50% 60% and 80% GC oligos were used indifferent experiments. The results, expressed as a graph of meltingtemperature vs GC content is shown as FIG. 7.

EXAMPLE 2

[0031] The different GC duplexes used in Example 1 were cloned into thevector polylinker of pUC19 plasmid as illustrated in FIG. 6. Thisplasmid was subjected to a polymerase chain reaction using vector primersites, the −47 and −48 sequencing primer sites. The PCR reactioncontained 1:40,000 dilution of SYBRGold™ reference dye. After PCR on theLightCycler™ the products were melted off as described in Example 1. Themelting temperature of the different amplicons vs the GC content isshown on the graph (FIG. 7).

1. A method of monitoring the temperature of a biochemical reaction,said method comprising effecting the reaction in the presence of afluorescently labelled temperature probe DNA sequence which comprises adouble stranded region which denatures at a predetermined temperature,the fluorescent label of said temperature probe sequence being arrangedso that a detectable signal occurs at the point at which denaturation ofthe said region takes place; and monitoring fluorescence from saidreaction mixture so as to determine when the said predeterminedtemperature has been reached.
 2. A method according to claim 1 whereinthe temperature probe DNA sequence comprises a labelled double strandedDNA sequence.
 3. A method according to claim 1 wherein the temperatureprobe DNA sequence comprises a single nucleic acid strand, and regionsof which hybridise together so as to form a loop or “hairpin” structure.4. A method according to any one of the preceding claims wherein thefluorescent label comprises an intercalating dye.
 5. A method accordingto claim 4 wherein the intercalating dye comprises SYBRGreen™ orSYBRGold™ or ethidium bromide.
 6. A method according to any one ofclaims 1 to 3 wherein the fluorescent label used in the method of theinvention may utilise fluorescence resonance transfer (FRET) as thebasis of the signal.
 7. A method according to claim 6 wherein thetemperature probe DNA sequence is provided with a reporter and aquencher alters the spatial relationship between the quencher andreporter molecules.
 8. A method according to claim 7 wherein thetemperature probe sequence is a single stranded sequence, where the endportions hybridise together and wherein the reporter molecule isattached in the region of either the 5′ or the 3′ end of the sequenceand the quencher molecule is attached at the opposite end.
 9. A methodaccording to claim 8 wherein the reporter and quencher molecules arelocated on different strands of a DNA temperature probe sequence suchthat on hybridisation of the strands, they are brought into closeproximity to each other.
 10. A method according to claim 9 wherein FRETis established between an intercalating dye and a quencher moleculearranged on a strand of the temperature probe sequence such that it canabsorb radiation from dye which is in close proximity on hybridisationof the strands.
 11. A method according to claim 7 wherein thetemperature probe DNA sequences comprises a first DNA strand having areporter molecule thereon, a second DNA strand having a quenchermolecule thereon, said first and second DNA strands being designed tohybridise to a third DNA strand such that the reporter and quenchermolecules are brought into close proximity with each other.
 12. A methodaccording to any one of the preceding claims wherein the length of thetemperature probe sequence is used to set the said predeterminedtemperature.
 13. A method according to any one of the preceding claimswherein the GC content of the temperature probe sequence is modified toobtain the desired predetermined temperature.
 14. A method according toany one of the preceding claims wherein the biochemical reaction is anamplification reaction.
 15. A method according to claim 14 wherein theamplification reaction is a polymerase chain reaction (PCR).
 16. Amethod according to claim 15 wherein the length of the temperature probesequence is similar to that of an amplicon of the PCR reaction.